JP2013205374A - Quantitative method of antigen sample - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for inexpensively and simply quantitating an antigen sample by using an immunochromatographic kit.SOLUTION: A quantitative method includes: a step which by using the immunochromatographic kit including an immunochromatographic carrier and successively arraying an antigen addition part for adding the antigen sample, a detection line on which a second antibody coupled with a complex of a first mark antibody and the antigen sample is immobilized and a comparison line on which the second antibody is immobilized on the carrier, develops the complex on the carrier from the antigen addition part up to the comparison line, and then measures first signal intensity in the detection line obtained by the mark and second signal intensity in the comparison line; a step for calculating a ratio of the first signal intensity to the sum of the first signal intensity and the second signal intensity; and a step for calculating the quantity of the antigen sample from the ratio.

Description

  The present invention relates to a method for quantifying an antigen sample.

  In recent years, simple immunochromatography kits have been used for influenza virus tests, pregnancy tests, and the like. This kit places importance on simplicity and can detect an antigen qualitatively. However, in order to obtain quantitativeness, an expensive detection device such as an immunochromatography reader is required. Thus, when processing the result obtained using the kit as image data, a contrivance has been made such as removing the influence of the background (background color) (see, for example, Patent Document 1).

JP 2000-338106 A

  An object of the present invention is to provide an inexpensive and simple method for quantifying an antigen sample using an immunochromatography kit.

  One embodiment of the present invention is a method for quantifying an antigen sample in an immunochromatography kit, the kit having an immunochromatography support, and an antigen addition unit for adding the antigen sample on the support A detection line on which a second antibody that binds to the complex of the first labeled antibody and the antigen sample is immobilized, and a control line on which the second antibody is immobilized, in this order, Measuring the first signal intensity in the detection line and the second signal intensity in the control line obtained by the labeling, after developing the carrier from the antigen addition part to the control line, Calculating the ratio of the signal intensity of 1 and the sum of the first signal intensity and the second signal intensity, and calculating the amount of the antigen sample from the ratio. No. In this quantification method, the ratio of the first signal intensity and the sum of the first signal intensity and the second signal intensity calculated using a plurality of antigens having the known antigen amount and the same antigen sample The amount of the antigen sample may be calculated using a standard curve that represents the relationship between the amount of the antigen and the ratio that has been created in advance. In addition, the quantification method further includes a step of obtaining image data of the kit after the complex is developed on the carrier to the control line, and in the image data, in pixels having a predetermined density or more in each signal. Each signal intensity may be measured by calculating the sum of the concentrations. The image data is preferably obtained using a scanner or a digital camera.

  According to the present invention, an antigen sample can be quantified inexpensively and easily using an immunochromatography kit.

It is a figure which shows an example of the kit for immunochromatography in one embodiment of this invention. In one Embodiment of this invention, it is the figure which graphed the signal obtained using the kit for immunochromatography. In one embodiment of the present invention, the ratio between the signal intensity in the test line obtained using the immunochromatography kit and the sum of the signal intensity in the test line and the signal intensity in the control line is quantified, and It is a figure showing a correlation. In one Embodiment of this invention, it is the figure which compared the result of the image-analysis method of this invention, and the conventional absorbance method.

  Hereinafter, embodiments of the present invention completed based on the above knowledge will be described in detail with reference to the accompanying drawings. The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for illustration or explanation, and the present invention is not limited to them. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

== Structure of immunochromatography kit ==
An example of an immunochromatography kit used in the antigen sample quantification method of the present invention is shown in FIG.

  The antigen to be quantified is not particularly limited as long as it can specifically bind to an antibody that can be used for the quantification of the present invention. For example, a high molecular weight compound such as a low molecular weight compound, sugar, protein, etc. Examples thereof include molecular compounds and metals.

  The immunochromatography kit 1 has an immunochromatography carrier 2, an antigen addition unit 3 for adding an antigen sample on the carrier, and a second binding to a complex of the first labeled antibody and the antigen sample. The detection line 4 to which the second antibody is immobilized and the control line 5 to which the second antibody is immobilized are arranged in this order. A reagent supply unit 6 for supplying the first labeled antibody may be provided between the antigen addition unit 3 and the detection line 4. As the material of the antigen adding portion 3, water-absorbing porous materials such as glass fiber, filter paper such as cellulose filter paper, polyurethane, polyacetate, cellulose acetate, nylon, and cotton cloth are used, and usually formed in a pad shape. The As a material for the reagent supply unit 6, a water-absorbing porous material such as glass fiber, filter paper, water-absorbing paper, felt-like fiber body, non-woven fabric, and absorbent cotton is used, and is usually formed in a pad shape. The carrier for immunochromatography 2 is not particularly limited as long as it is a porous membrane used for immunochromatography. For example, nitrocellulose membrane, cellulose membrane, acetylcellulose membrane, polysulfone membrane, polyethersulfone membrane, nylon membrane, glass fiber , Non-woven fabric, cloth or yarn. Moreover, the water absorption pad 7 which consists of a water absorbing material may be provided in the downstream of the expansion | deployment direction of the support | carrier 1 for immunochromatography. Examples of the water absorbing material include nitrocellulose, non-woven fabric, cloth, cellulose acetate, and paper.

  Examples of the labeling substance bound to the first labeling antibody include gold colloid, enzyme, fluorescent substance, and the like, but gold colloid is preferable in terms of simplicity. Examples of enzymes include peroxidase, alkaline phosphatase, glucose oxidase and the like, and these can be used alone or in admixture of two or more.

  The first labeled antibody is not particularly limited as long as it binds to the antigen sample, but the higher the specificity of the binding, the better. The second antibody is not particularly limited as long as it binds to the complex of the first labeled antibody and the antigen sample, but the higher the specificity of the binding, the more preferable the antibody is to the antigen sample. May be the same as one antibody, but should not interfere with the detection of the label. The second antibody is immobilized on the detection line 4 and the control line 5 of the immunochromatography carrier 2, but is preferably immobilized in a line perpendicular to the direction in which the antigen sample flows. The amount of the second antibody immobilized on the detection line 4 is not particularly limited, but is preferably an amount that can capture the first labeled antibody bound to the antigen sample and detect color development by the label. The amount of the second antibody immobilized on the control line 5 is preferably an amount that can capture all of the first labeled antibody that has not been captured by the detection line.

== Method for quantifying antigen sample ==
An example of a method for quantifying an antigen sample will be described in detail using the immunochromatography kit 1 described above.

  First, when the reagent supply unit 6 is not provided, a plurality of known amounts of the same antigen as the antigen sample are mixed with the first labeled antibody, and the antigen and the first labeled antibody are combined to produce a complex thereof. To do. This complex is added to the antigen addition part 3 on the immunochromatography kit 1. The complex of the antigen and the first labeled antibody develops on the immunochromatographic carrier 2 up to the control line 5 by capillary action using the liquid component containing the complex as a developing solution. You may exceed. Providing the water absorbing pad 7 helps to increase the deployment speed.

  When the reagent supply unit 6 is provided, the first labeled antibody is previously contained in the reagent supply unit 6 in a state where it can be moved without being immobilized. When an antigen is added to the antigen addition unit 3, the antigen moves with the development of the solution, a complex is formed in the reagent supply unit 6, and moves with the development of the solution.

  When the developing solution passes through the detection line 4 and the control line 5, the complex in the developing solution is captured by the second antibody immobilized on each line. Since a large amount of antibody is immobilized on the control line 5, virtually all of the first labeled antibody that has not been captured by the detection line 4 is captured.

  Thereafter, when the label is an enzyme, a substrate is added to the detection line 4 and the control line 5 to develop a signal. It is preferable to drop the substrate solution so that the color can be easily developed. When the label is a colloidal gold, it is concentrated in the detection line 4 and the control line 5 and visualized.

  The first signal intensity in the detection line 4 and the second signal intensity in the control line 5 thus obtained are measured. An immunochromatography reader may be used for the measurement, but it is convenient to carry out as follows. First, image data of each signal is created using a scanner or a digital camera. The image data is preferably in gray scale because the accuracy of signal intensity measurement is enhanced by extracting colors in a specific range of wavelengths. Then, in the image data, the sum of the density is calculated for pixels having a predetermined density or more in each signal, and the intensity of each signal is measured. For example, using signal analysis software (such as Scion image or NIH Image), the signal intensity may be graphed as shown in FIG. 2, and the area of the mountain at a certain level or higher may be measured. Then, the sum of the first signal intensity and the second signal intensity is calculated, the ratio of the first signal intensity and the sum is calculated, and the correlation between the used antigen amount and the calculated ratio is graphed as shown in FIG. Into a standard curve.

  Next, for the antigen sample to be quantified, the ratio of the first signal intensity and the sum of the first signal intensity and the second signal intensity is calculated in the same manner as when a standard curve is prepared. Then, the concentration of the antigen sample can be calculated using a standard curve prepared in advance.

  First, an immunochromatography kit 1 for detecting the mussel adhesion stage larvae of 7.7 cm × 2.0 cm (thickness 0.5 cm) was prepared, and 1.5 cm, 4.0 cm, and 4.5 cm sites were formed from one end. Antigen sample dripping site 3, test line 4 (corresponding to the above detection line), control line 5 (corresponding to the above control line), and test line 4 and control line 5 each have 0.75 μg of anti-mussel mussel adhesion stage larvae monoclonal antibody Then, 0.3 μg of mouse anti-IgG antibody (Mouse, Goat-Poly) was applied to the membrane linearly with a line applicator (BioDot, Quantum Design, Japan) and dried. Further, 0.61 μg (as a weight not including gold colloid) of the colloidal gold labeled anti-mussel larvae monoclonal antibody labeled as colloidal gold (as a weight not including the gold colloid) was uniformly applied and dried.

  On the other hand, the mussel larvae 1, 10, and 100 individuals obtained in captivity were placed in a 15 mL centrifuge tube containing 1 mL of seawater and crushed by a homogenizer. After standing for 5 minutes, 0.1 mL of the supernatant was used as an antigen sample. As a control, 0.1 mL of seawater containing no larvae extract was used. These antigen samples were dropped on the antigen sample dropping pad 3, and the carrier 2 was developed to the test line. Thereafter, the detection frame portion of kit 1 from which a signal was obtained was photographed with a scanner (canoscan 9950F, manufactured by Canon Inc.) (image resolution 1200 dpi, gray scale) to produce an image file (tiff file). Thereafter, the signal intensity was graphed with image analysis software (Socion Image). An example thereof is shown in FIGS. In B, a signal that cannot be observed visually (portion surrounded by a dotted line) is detected, indicating that the sensitivity of the present method is high.

  When each antigen sample was analyzed, signal intensities as shown in Table 1 were obtained (cont: control line signal intensity, test: test line signal intensity). The results are graphed in FIG.

On the other hand, the absorbance of the test line alone was measured for the kit from which the signal was obtained with an immunochromatographic reader (absorbance method), and the results are plotted in FIG. 4 together with the results of the image analysis method of the present invention. In addition, the signal intensity by the image analysis method of the present invention described in Table 1 and FIG. 4 is a value obtained by multiplying the raw data by 270 so that it can be directly compared with the value of the immunochromatography reader.

  If the standard curve that can be prepared in this way is used, the target antigen sample can be quantified easily.

1 Immunochromatography kit 2 Immunochromatography carrier 3 Antigen sample dropping pad (antigen addition part)
4 Test line (detection line)
5 Control line (control line)
6 Reagent pad (reagent supply unit)
7 Water absorption pad

Claims (4)

A method for quantifying an antigen sample in an immunochromatography kit, comprising:
The kit has an immunochromatography carrier, and an antigen addition part for adding an antigen sample and a second antibody that binds to a complex of the first labeled antibody and the antigen sample are immobilized on the carrier. Detection line, and a control line to which the second antibody is immobilized in this order,
The step of measuring the first signal intensity in the detection line and the second signal intensity in the control line obtained by the labeling after the complex is developed on the carrier from the antigen addition part to the control line When,
Calculating a ratio of the first signal intensity and the sum of the first signal intensity and the second signal intensity;
Calculating the amount of the antigen sample from the ratio;
A quantitative method comprising:   Using a ratio of the first signal intensity and the sum of the first signal intensity and the second signal intensity calculated using the same plurality of antigens as the antigen sample having a known antigen amount, The quantification method according to claim 1, wherein the amount of the antigen sample is calculated using a prepared standard curve representing the relationship between the amount of the antigen and the ratio. Further comprising obtaining image data of the kit after the complex has developed on the carrier to the control line;
3. The method according to claim 1, wherein in each of the image data, each signal intensity is measured by calculating a sum of densities in pixels that are equal to or higher than a predetermined density in each signal.   The method according to claim 3, wherein the image data is obtained using a scanner or a digital camera.